S100B和S100A1可作為抗癌藥物之醫藥用蛋白｜國立清華大學博碩士論文庫

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研究生：	東郭旺 Dowarha, Deepu
論文名稱：	S100B和S100A1可作為抗癌藥物之醫藥用蛋白 S100B and S100A1 are the pharmaceutical proteins against cancer disease
指導教授：	余靖 Yu, Chin
口試委員:	蘇士哲 Su, Shih-Che 莊偉哲 Chuang, Woei-Jer 陳金榜 Chen, Chin-Pan 鄒瑞煌 Chou, Ruey-Hwang
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	139
中文關鍵詞：	醫藥用蛋白
外文關鍵詞：	Pharmaceutical proteins
相關次數：	點閱：2 下載：0
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目前已知S100蛋白質家族具有許多細胞內和細胞外功能，例如鈣穩態，細胞增殖和分化，凋亡，轉錄，組織發育和修復。近年來，S100蛋白引起了研究界的關注，因為S100蛋白與多種導致腫瘤的過程有關，其表達模式的改變與多種人類癌症有關。RAGE（晚期糖基化終產物的受體）是S100蛋白的一般受體之一，S100蛋白也與癌症控制蛋白p53和MDM2（鼠雙分2）有關。
S100，RAGE，p53和MDM2的蛋白質與蛋白質交互作用研究將有助於我們理解它們在癌症控制和生長中的作用。S100蛋白及其靶分子之間形成的大分子復合物結構將能提供有用的數據，這些數據可為尋求抗癌特異性治療分子的藥物研究提供支持。為了理解這一點，我們研究了蛋白質-蛋白質相互作用研究，包括S100A11，S100B，RAGE，S100A1，p53和MDM2蛋白質。
在研究項目1（第二章）中，我們通過1H-15N HSQC-NMR（heteronuclear single-quantum correlation-NMR）滴定法研究了S100A11和S100B蛋白之間的相互作用。然後，我們利用HADDOCK程序構建了S100A11–S100B異二聚體複合物，然後將其與之前報導的S100A11–RAGE V域複合物疊加。疊圖數據證明，S100B可能阻礙S100A11與RAGE V域的結合界面區域。此外，WST-1（水溶性四唑-1）測定法可提供這些蛋白質在體外癌症模型中作用的功能性讀數。我們的研究證實，S100B拮抗劑的改良可以在S100和RAGE的人類疾病治療中扮演重要的角色。

在研究項目2（第三章）中，我們將工作重點放在可能干擾p53-MDM2相互作用的拮抗劑的開發上，因為據推測，刺激wild-type p53活性的有效策略可能需要打斷p53-MDM2相互作用，從而恢復具有wild-type p53的腫瘤中p53腫瘤抑制能力。目前已知，S100A1蛋白與MDM2和p53蛋白的N末端結構域有相互作用，我們對於當S100A1蛋白與MDM2和p53蛋白相互作用時， S100A1的界面區域的研究十分感興趣，此介面區物將作為結構的藥物設計方法的一部分，以發展出合適的拮抗劑干擾p53-MDM2交互作用。我們應用核磁共振光譜研究了S100A1與MDM2和p53的N末端結構域之間的結合界面。使用NMR和HADDOCK方法進行的數據分析顯示出S100A1片段（17個殘基）可能成功阻止p53-MDM2相互作用。為了檢驗癌細胞系中的假設，我們合成了源自S100A1蛋白的17個殘基的肽，並將其連接到可穿透細胞的HIV-TAT肽上，並將其命名為Peptide 1。來自HSQC-NMR competitive bingding實驗，WST-1分析，蛋白質印跡和細胞週期分析的合作數據支持了我們的假設，並表明peptide 1可以成功干擾p53-MDM2相互作用並激活正常的p53功能導致癌細胞的細胞週期停滯和凋亡細胞死亡。此證明了針對癌症生長，開發更多訂製藥物分子的可能性。
總體而言，通過我們的研究工作，我們更理解分子層級上與S100A11，S100B，RAGE，S100A1，p53和MDM2蛋白質有關的蛋白質-蛋白質和/或蛋白質-配體之間的交互作用，並為他們在訂製的抗癌藥物分子的開發與應用做出了貢獻。

S100 family of proteins are known to possess numerous intracellular and extracellular functions such as calcium homeostasis, cell proliferation and differentiation, apoptosis, transcription, tissue development, and repair. In recent times, S100 proteins have attracted the focus of the research community as S100 proteins are found to relate with various tumorigenic processes and their altered expression patterns are associated with multiple human cancers. One of the general receptors of S100 proteins is RAGE (Receptors for advanced glycation end products) and S100 proteins are also found to associate with the cancer controlling proteins p53 and MDM2 (Murine double minute 2). Protein-protein interaction studies involving the S100, RAGE, p53, and MDM2 will elucidate important aspects which will be helpful to understand their role in cancer control and/or progression. The structural insights into the macromolecular complex formation amongst S100 proteins with its target molecules will provide useful data which will be supportive in the pharmaceutical research for the quest of specific therapeutic molecule/s against cancer. To comprehend this, we studied the protein-protein interaction studies which included S100A11, S100B, RAGE, S100A1, p53, and MDM2 proteins.
In research project 1 (Chapter II), we studied the interactions amongst S100A11 and S100B proteins by employing the 1H–15N HSQC-NMR (heteronuclear single-quantum correlation-NMR) titrations. Then, we utilized the HADDOCK program to construct the S100A11–S100B heterodimer complex following which it was superimposed with the S100A11–RAGE V domain complex reported earlier. The overlay data exploration revealed that the S100B could obstruct the binding interface region of S100A11 and the RAGE V domain. Furthermore, WST-1 (water-soluble tetrazolium-1) assay provided a functional read-out of the effects of these proteins in an in vitro cancer model. Our study establishes that the improvement of a S100B antagonist could perform a vital part in the treatment of S100- and RAGE-dependent human diseases.
In research project 2 (Chapter III), we focused our work towards the development of antagonist that could interfere with the p53-MDM2 interaction, as it was postulated that the effective strategy to stimulate the activity of wild type p53 may require the breakdown of p53-MDM2 interaction, restoring the p53 tumor suppressor capability in tumors with wild-type p53. The S100A1 protein was reported to interact with the N-terminal domain of MDM2 and p53 protein and therefore attracted our interest to study the interface region of S100A1 when interacted with MDM2 and p53 protein as a part of structure-based drug design approach for the development of suitable antagonist interfering with the p53-MDM2 interaction. We applied NMR spectroscopy to study the binding interface amongst the S100A1 and N-terminal domain of MDM2 and p53. The data analysis using NMR and HADDOCK methods showed the possibility of S100A1 segment (17 residues) that could block the p53-MDM2 interaction successfully. To test the hypothesis in the cancer cell line, we synthesized the 17-residue peptide derived from the S100A1 protein and attached it to the cell-penetrating HIV-TAT peptide and named it as Peptide 1. The collaborative data from the HSQC-NMR competitive binding experiment, WST-1 assay, western blotting, and the cell cycle analysis supported our assumption and showed that the Peptide 1 could successfully interfere with the p53-MDM2 interaction and could activate the normal p53 functions, leading to cell cycle arrest and apoptotic cell death in cancer cells. This information shows the possibility for the development of more customized drug molecules against the cancer development.
Overall, through our research work we have contributed towards the understanding of protein-protein and/or protein-ligand interactions at a molecular level pertaining to the S100A11, S100B, RAGE, S100A1, p53, and MDM2 proteins, and their application in the development of customized drug molecule against cancer.

Table of contents
Abstract in Chinese………………………………………………………I
Abstract in English………………………………………………………III
Acknowledgement……………………………………………………………VI
Table of Contents…………………………………………………………IX
List of figures……………………………………………………………XIV
Abbreviations………………………………………………………………XXVI

Chapter - I
Introduction
1.1. Introduction ………………………………………………………………2
1.2. S100 family of Proteins…………………………………………………9
1.3. Receptor for advanced glycation end products (RAGE)……………11
1.4. Tumor protein 53 (TP53 or p53)…………………………………….…13
1.5. Murine double minute 2 (MDM2)…………………………………………15
1.6. Protein extraction and purification…………………………………18
1.7. Reverse-phase high-performance liquid chromatography (RP-HPLC).21
1.8. Protein analysis……………………………………………………………23
1.9. Protein-ligand interactions…  ………………………………………24
1.10. NMR spectroscopy to map protein-protein interaction in solution………………..............................................30
1.11. Chemical shift perturbation to demonstrate protein-ligand binding………………...............................................32
1.12. HADDOCK- a data driven molecular docking tool……………………………........................................37
1.13. WST-1 assay over MTT assay…………………………………………..41

Chapter - II
S100B as an antagonist to interfere with the interface area flanked by S100A11 and RAGE V domain
2.1. Introduction………………………………………………………………………44
2.2. Materials and Methods……………………………………………………46
2.2.1. Materials……………………………………………………………………….…46
2.2.2. Expression, Labeling, and Purification…………………………46
2.2.3. HSQC- NMR Titration Experiments…………………………………55
2.2.4. Molecular Docking……………………………………………………56
2.2.5. Cell Proliferation Assay……………………………………………57
2.3. Results and Discussion…………………………………………………57
2.3.1. The binding interface (S100A11/S100B) region in S100A11…………………….........................................57
2.3.2. The binding interface (S100A11/S100B) in S100B…………………………..…….................................60
2.3.3. The fundamental depiction of the S100A11-S100B complex……………………….......................................62
2.3.4. Dissociation constant………………………………………………65
2.3.5. WST-1 assay……………………………………………………………69
2.3.6. S100B inhibits the S100A11-RAGE V domain……………………70
2.4. Conclusion………………………………………………………………72

Chapter - III
S100A1 blocks the interaction between p53 and mdm2 and decreases cell proliferation activity
3.1. Introduction………………………………………………………………………75
3.2. Materials and methods……………………………………………………77
3.2.1 Materials………………………………………………………………………….77
3.2.2. Expression, Labeling, and Purification………………………….78
3.2.3. Peptide synthesis………………………………………………………95
3.2.4. HSQC- NMR titration experiments……………………………………95
3.2.5. Molecular docking……………………………………………………..96
3.2.6. HSQC- NMR competitive binding experiment………….……………96
3.2.7. Fluorescence experiment………………………………………………97
3.2.8. Circular dichroism (CD) spectroscopy…………………………….98
3.2.9. Cell proliferation assay…………………………………………….98
3.2.10. Western blotting……………………………………………………..98
3.2.11. Cell cycle analysis………………………………………………….99
3.3. Results and discussion………………………………………………….99
3.3.1. Interaction amongst labeled S100A1 and unlabeled MDM2………………………............................................99
3.3.2. Interaction amongst unlabeled S100A1 and labeled MDM2…………………….............................................101
3.3.3. Illustration of the S100A1−MDM2 complex……………………….103
3.3.4. Overlapping of S100A1-MDM2 complex with p53 (peptide)-MDM2 complex…………………………………………………………………………..106
3.3.5. S100A1 could interfere with the interaction amongst p53 and MDMX……..….....................................................107
3.3.6. Interaction amongst unlabeled S100A1 and labeled p53 (1-73)…………….….................................................108
3.3.7. Interaction amongst labeled S100A1 and unlabeled p53 (1-73) and comparison of S100A1-p53 (1-73) HSQC-NMR data with S100A1-MDM2 HSQC-NMR data……....................................................110
3.3.8. S100A1 derived peptide construction……………………………111
3.3.9. CD spectra……………………………………………………………113
3.3.10. Fluorescence experiment…………………………………………114
3.3.11. Competitive binding experiment by NMR………………………116
3.3.12. WST-1 assay…………………………………………………………118
3.3.13. Western Blotting………………………………………………….120
3.3.14. Cell cycle analysis………………………………………………121
3.4. Conclusion……………………………………………………………  122













List of figures
Chapter- I
Figure 1.1. Schematic illustration of bacterial cell E. coli……….2
Figure 1.2. Schematic depiction of eukaryotic cell…………………….3
Figure 1.3. The diagrammatic representation of flow of genetic information…..……….…...........................................4
Figure 1.4. The diagrammatic representation of secretary pathway of protein in a eukaryotic cell………………………………………………………………............….5
Figure 1.5. (a) Illustration of the basic amino acid structure and (b) the association of two amino acid subunits leads to the formation of peptide bond…………………..……….............................….7
Figure 1.6. Figure showing the four structural stages of a protein structure……….……..............................................8
Figure 1.7. Schematic and cartoon illustration of S100 protein basic structure. (a) L1 and L2 are the Ca2+ binding loop flanked by the α-helices HI to HIV. Hinge region, H, connects the HII and HIII.20 (b) The cartoon displaying apo-S10011 dimeric form with Ca2+ binding loops in red color and α-helices labelled in both chains of the apo-S100A11 protein…………………………………………………………………...…...10
Figure 1.8. Calcium induced conformational changes in calcium-free (Apo) and calcium-bound S100A11 protein………………..………...…..11
Figure 1.9. Schematic representation of RAGE protein…………………12
Figure 1.10. S100 Protein binding to RAGE triggers various RAGE mediated cellular signaling. Few S100 proteins, such as, S100B, S100P, S100A8/A9, S10012, and S100A14 binds to RAGE and activates NF-KB, MAPK, and PI-3K/AKT signaling pathways thereby regulating cellular processes including inflammation and cancer. S100A6 initiates the cell apoptosis by activating JNK pathway and restricts cell proliferation…………………………………………………...……………..13
Figure 1.11. Schematic diagram of p53 indicating various functional domains……...……................................................14
Figure 1.12. Graphic illustration of MDM2 indicating various functional domains……..........................................…16
Figure 1.13. Schematic diagram of the p53-mdm2 auto-regulatory feedback loop. P53 positively regulates MDM2 by controlling MDM2 gene expression and MDM2 negatively regulates p53 biological role by three possible means………………………………….…......................17
Figure 1.14. Schematic representation of steps involved in protein purification after its expressio…………………………………...…….21
Figure 1.15. Schematic representation of HPLC system basic components…………............................................……23
Figure 1.16. Schematic basic illustration of mass spectrometer components and data flow……………………………………………...…...24
Figure 1.17. The schematic representation of all the three binding models. (a) Protein and ligand are rigid and binds to each other as interface region is complementary to each other as a feature of lock and key model. (b) Ligand binding induces a conformational change in protein binding site for enhanced fitting as the protein binding interface is quite flexible and represents induced fit model. (c) In conformational selection model, ligand binds to the best fitting conformational state of the protein amongst a population of varied protein states……………………………………………………………………….…...27
Figure 1.18. Superimposition of the 2D spectra of labeled MDM2 (red) and in complex with Amx drug in 1:1 (blue) and 1:2 (yellow) ratio. Peak shift is observed as a result of ligand binding (Amx) to protein (MDM2) and is highlighted in dotted white color………......……...34
Figure 1.19. Illustration of fast and slow exchange system. Left panel shows the fast exchange, where peaks move from free (blue) to bound state (red) freely with some broadening at midway locations. Right panel shows the slow exchange, where the free peak (blue) intensity decreases during the course of titration and the bound peak (red) intensity increases……………………………………….……………….….36
Figure 1.20. The online HADDOCK server showing Easy interface mode…..…………..................................................40
Figure 1.21. The conversion of MTT to Formazan indicative of living cells………..…...................................................41
Figure 1.22. The conversion of WST-1 to Formazan indicative of living cells……….......................................................42

Chapter- II
Figure 2.1. Human S100A11 (wild type) clone construct. (a) The map of pET-20b (+) vector displaying various cloning sites. NdeI and/or XhoI restriction sites was used for the insertion of S100A11 protein sequence. (b) Amino acid sequence of S100A11 protein showing one letter amino acid code for each amino acid…………………………….47
Figure 2.2. Chromatogram displaying (a) S100A11 purification via HIC column followed by (b) SDS-PAGE………………….……………………..49
Figure 2.3. Elute fractions (E1 and E2), from HIC column purification, were mixed, concentrated and purified further via HPLC column. (a) HPLC chromatogram displaying pure S100A11 peak which is identified and verified for purity with (b) SDS-PAGE followed by (c) ESI-MS mass confirmation……………………………………......………..…………..50
Figure 2.4. Amino acid sequence of S100B protein showing one letter amino acid code for each amino acid……………………………………51
Figure 2.5. Chromatogram displaying S100B purification via Q column and the inset shows the SDS-PAGE indicating the E1 and E2 elute fractions contain S100B protein...........................................................52
Figure 2.6. Elute fractions (E1 and E2), from Q column purification, were mixed, concentrated and purified further via SEC column. Chromatogram illustrating S100B purification via SEC column and the inset shows the SDS-PAGE displaying the fractions 1 to 9 tested for S100B protein. Fractions 6 and 7 contained least impure S100B protein…………………………………………………………………………...53
Figure 2.7. Elute fractions (6 and 7), after SEC column purification, were mixed, concentrated and purified further via HPLC column. (a) HPLC chromatogram displaying pure S100B peak which is identified and verified for purity with (b) SDS-PAGE followed by (c) ESI-MS mass confirmation…………………………….………………........……….….54
Figure 2.8. Diagrammatic representation of steps involved in the dialysis process…….............................................56
Figure 2.9. Analysis of the binding interface (S100A11/S100B) reion in S100A11. (a) 1H-15N HSQC spectra overlay of free 15N S100A11 (red) and 15N S100A11 binding to unlabeled S100B (blue). Cross-peaks illustrating changes in intensities are shown in boxes. Horizontal lines connect the NH2 side chains. (b) Cross-peak intensity plot (I/I0) where (I) denotes cross peak intensity of S100A11-S100B complex and (I0) indicates the early intensity of free S100A11 versus a number of amino-acid residues (1-105) illustrated as a bar diagram. (c) A picture illustration of the S100A11 monomer with residues exhibiting decreases in cross-peak signals distinguished by the cyan color………………………………………….….........................59
Figure 2.10. Analysis of the binding interface (S100A11/S100B) region in S100B. (a) 1H-15N HSQC spectra overlay of free 15N S100B (red) and 15N S100B binding to unlabeled S100A11 (blue). Cross-peaks showing changes in intensities are shown in boxes. (b) Cartoon representation of S100B monomer with residues exhibiting decreases in cross-peak signals are shown in yellow…………………………………………………62
Figure 2.11. (a) Cartoon representing S100A11-S100B complex created with HADDOCK program. S100A11 is depicted in red and the S100B in blue color. Residues near the interaction sites are illustrated in cyan (from the S100A11 side) and yellow (from S100B side) respectively. (b) List of S100A11 and S100B residues as constraints for HADDOCK program……………………………..……………………………………………..63
Figure 2.12. The Ramachandran plot. The online PROCHECK program was used to test the structural stereochemistry of the complex shown in figure 2.11. The Ramachandran plot disclosed the occurrence of 82.1% residues in the maximum favoured regions, 13.4% in additionally allowed areas, 2.8% in allowed sector, and 1.7% in the disallowed zone………………………………………………………………………………..64
Figure 2.13. 1H- 15N HSQC spectra of free 15N S100A11 (red)………65
Figure 2.14. 1H- 15N HSQC spectra overlay of free 15N S100A11 (red) and 15N S100A11 homodimer + S100B homodimer mixture (blue)…………..66
Figure 2.15. 1H- 15N HSQC spectra overlay of free 15N S100A11 (red) and 15N S100A11- S100B heterodimer (green)……………………........67
Figure 2.16. Dissociation constant. The Kd calculated by the designated residues detected in 15N S100A11 HSQC titrations with S100B (yellow) and vice-versa (brown). The overall average Kd is about 2.65 μM…………………………………………………..…………...............68
Figure 2.17. WST-1 assay analysis. The SW480 cells were treated with 100 nM S100 A11 (dark blue), 100 nM S100B (light blue), and 100 nM S100A11-S100B heterodimer (green). The proportional cell counts after subsequent treatment with S100A11 and S100B are depicted as fold inductions with serum-free media alone as the control (red)………………………………………………………………..…….……..70
Figure 2.18. S100B interfering S100A11-RAGE V domain. (a) The complex model of S100A11-RAGE V domain constructed via HADDOCK. (b) Superimposition of the complex between S100A11 (red) and the RAGE V domain (green), with the complex amid S100A11 (red) and S100B (blue). (c) The magnified depiction of the clear and significant hindrance to S100A11-RAGE V domain associated with the S100B protein…………………………………………………………………………...72

Chapter- III
Figure 3.1. The picture illustration of pET28-p53 (1-73) vector…79
Figure 3.2. His tag fused p53 (1-73) protein (fusion protein) showing the amino acid sequence (a) before enzyme digestion with the site for thrombin cutting, and (b) sequence after enzyme digestion…………79
Figure 3.3. Chromatogram displaying p53-TAD (1-73) purification via NiNATA Superflow resin column and the inset shows the SDS-PAGE indicating various fractions. S represents the crude sample loaded onto the column; LB, WB, and EB indicate the elute fractions collected by employing lysis buffer, wash buffer, and the elution buffer. EB fraction contained the p53-Histidine tag fusion protein (10.13 kDa) which comes above the 15 kDa band before enzyme digestion…………81
Figure 3.4. Following enzyme digestion with Thrombin, digested fusion protein sample was loaded onto the Superdex 75 (SEC) column and the inset shows SDS-PAGE displaying the fractions (1 to 7) collected after enzyme digestion and cleaved p53 (1-73) protein (8.6 kDa) is observed (fraction, 5, 6, and 7) below the 15 kDa band, though fraction 7 has less protein concentration…………………………………………………………………...82
Figure 3.5. Following SEC purification, fraction consisting pure protein was subjected to the ESI-MS analysis for the confirmation of cleaved p53 (1-73) protein having calculated mass of 8617.53 kDa…………………………………………………………….......…………83
Figure 3.6. The picture illustration of pGEX6P2-MDM2 (17-125) vector…….………….............................................84
Figure 3.7. GST tag fused MDM2 (17-125) protein (fusion protein) showing the amino acid sequence (a) before enzyme digestion with the site for PreScission Protease enzyme cleavage, and (b) sequence after enzyme digestion………………………………..……....................85
Figure 3.8. Chromatogram displaying GST-MDM2 fusion protein purification via GST column and the inset shows the SDS-PAGE indicating the E1 and E2 elute fractions containing the fusion protein…………………………………………..……………..........…….86
Figure 3.9. Chromatogram displaying the purification of cleaved MDM2 protein via GST column after enzyme digestion. The inset shows the SDS-PAGE where, S represents the crude MDM2-GST tag fusion protein, F1 is the flow and E1 is the elute collected before enzyme digestion, AED represents the E1 sample incubated with the PreScission protease enzyme for the 16 hours’ enzyme digestion, F2 represents the cleaved MDM2 protein (12.9 kDa) collected in flow after enzyme digestion, E3 represents the mixture of any remained fusion protein (39.3 kDa) together with cleaved GST tag (26.4 kDa), and M indicates the protein marker…………………………………………………………………………….87
Figure 3.10. (a) HPLC chromatogram displaying cleaved MDM2 peak at number 4……......................................................88
Figure 3.10. (b) SDS-PAGE displaying the presence of cleaved MDM2 protein in fraction 4 corresponding to peak 4 of HPLC profile……88
Figure 3.10. (c) Cleaved MDM2 protein in fraction 4 corresponding to peak 4 of HPLC profile was confirmed by ESI-MS analysis……………89
Figure 3.11. Human S100A1 clone construct. (a) The map of pET-20b (+) vector displaying various cloning sites. NdeI and/or XhoI restriction sites was used for the insertion of S100A1 protein sequence. (b) Amino acid sequence of S100A1 protein showing one letter amino acid code for each amino acid…………………………………………….……….........90
Figure 3.12. Purification of S100A1 protein. (a) Chromatogram displaying S100A1 purification via Q-column. Elute 1 consisting S100A1 protein is collected, concentrated, and loaded on to the HIC column. (b) Chromatogram displaying S100A1 purification via HIC-column and the inset shows the SDS-PAGE indicating various fractions. S represents the crude sample, F1 and E1 indicates the flow through and elute fractions collected by employing Q- column. F2 and E2 indicates the flow through and elute fractions collected by employing HIC- column…………………………………………………………..…….........92
Figure 3.12. (c) E2 fraction collected by employing HIC-column is concentrated and loaded on to the SEC column. Fractions 4,5, and 6 contained the S100A1 protein mostly…………………………………...93
Figure 3.13. (a) HPLC chromatogram shows the S100A1 purification profile and the inset SDS-PAGE illustrates the pure S100A1 peak (peak 2) which is identified and verified for purity, followed by (b) ESI-MS mass confirmation……………………………………….................94
Figure 3.14. Exploration of the connected interface (S100A1/MDM2) section in S100A1. (a) The 1H−15N HSQC spectra overlay of free 15N S100A1 (red) and in complex with unlabelled MDM2 (blue). Cross-peaks displaying variation in their magnitude are highlighted in yellow. Residues were assigned as published earlier.226 (b) A picture illustration of the S100A1 protein with residues demonstrating a change in cross-peak signals portrayed by the red color…………101
Figure 3.15. Exploration of the connected interface (S100A1/MDM2) section in MDM2. (a) The 1H−15N HSQC spectra overlay of free 15N MDM2 (red) and in complex with unlabelled S100A1 (yellow). Cross-peaks illustrating variation in their magnitude is highlighted in blue. Residues were assigned as published earlier.229 (b) A picture showing the MDM2 protein with residues displaying change in cross-peak signs, which are characterized by the blue color……………………………..102
Figure 3.16. (a) Animation displaying S100A1−MDM2 complex generated via the HADDOCK program. S100A1 is painted in green and MDM2 in yellow color. Residues neighboring the interface regions are indicated in red (from the S100A1 side) and blue (from MDM2 side), respectively. (b) List of S100A1 and MDM2 residues as constraints for HADDOCK program.…………………...………………………...……………………..104
Figure 3.17. The Ramachandran Plot. The online PROCHECK program was used to test the structural stereochemistry of the complex shown in figure 3.16. The Ramachandran plot disclosed the occurrence of 93.2% residues in the maximum favored regions, 5.6% in additionally allowed areas, 1.2% in allowed sector, and 0.0% in the disallowed zone…………………………………………………………...………………….105
Figure 3.18. S100A1 interferes with the p53-MDM2 complex formation. (a) The complex model of the S100A1−MDM2 protein generated using the HADDOCK program. (b) The reported complex model of the p53 peptide−MDM2 domain obtained via the X-ray diffraction technique. (c) Overlay of the complex amid S100A1 (green) and the MDM2 domain (yellow), to the complex amid p53 peptide (blue) and MDM2 (yellow) showing S100A1 interference to p53-MDM2 complex………………………106
Figure 3.19. S100A1 can interfere with the interaction between p53 and MDM2/MDMX. (a) A reported complex model of p53 peptide and MDM2 N-terminal domain (PDB ID- 1YCR). (b) A reported complex model of p53 peptide and MDMX N-terminal domain (PDB ID- 3DAC). (c) A complex model of S100A1 and MDM2 N-terminal domain generated via the HADDOCK program. (d) Overlapping of the complex models indicated in a, b, and c showed possible interference of S100A1 between p53 peptide and MDM2/MDMX N-terminal domain…………………………………………………108
Figure 3.20. The 1H−15N HSQC spectra overlay of free 15N p53 (1-73) (red) and in complex with unlabelled S100A1 (yellow). Cross-peaks proving deviations in intensities are highlighted in yellow. Residues were assigned as published earlier……………………...............110
Figure 3.21. Exploration of the connected interface (S100A1/p53 (1-73)) section in S100A1. (a) The 1H-15N HSQC spectra overlay of free 15N S100A1 (red) and in complex with unlabelled p53 (1-73) (blue). Cross-peaks displaying variation in their magnitude are highlighted in yellow. (b) Table illustrating a list of the affected residues when 15N S100A1 interacts with the unlabeled p53 (1-73) and unlabeled MDM2 and also showing the common interacting residues……………………111
Figure 3.22. S100A1 complete sequence indicating highlighted region selected for peptide construction, attaching it to HIV-TAT peptide forming Peptide 1 and its scramble arrangement in Peptide 2……113
Figure 3.23. Far-UV CD spectra of (a) S100A1 protein and (b) Peptide 1……………......................................................114
Figure 3.24. The p53-MDM2 interaction was monitored via fluorescence spectroscopy. (a) Changes in the fluorescence emission spectra of p53 upon the addition of increasing concentration of MDM2 in the micro-molar range was observed. (b)  Relative intensity versus the MDM2 total concentration………………………………………..………….115
Figure 3.25. The p53-Peptide 1 interaction was monitored via fluorescence spectroscopy. (a) Changes in the fluorescence emission spectra of p53 upon the addition of increasing concentration of Peptide 1 in the micro-molar range was observed. (b)  Relative intensity versus the Peptide 1 total concentration………………………………………..……….........116
Figure 3.26. Competitive binding experiment. (a) The 1H-15N HSQC spectra of 15N MDM2 in complex with unlabeled Peptide 1 (red) in a 1:1 ratio. (b) The 1H-15N HSQC spectra of 15N MDM2 in complex with unlabeled p53 (blue) in a 1:1 ratio. To the same mixture, we added the Peptide 1 in a 1:1 ratio and obtained the spectrum as shown in (c). Then the spectra obtained in (b) and (c) is overlapped to the spectra indicated in (a) and the resulting overlapped spectra are shown in (d)……………………………………………….…….....................117
Figure 3.27. Analysis of WST-1 assay. The (a) MCF-7 and (b) AGS cells were treated with 5, 10, and 20 µM concentrations of Peptide 1 (blue) and Peptide 2 (green). The comparative cell counts after successive treatment with Peptides 1 and 2 are shown as fold inductions with the control (red) as serum-free media alone……………………………..…119
Figure 3.28. The MCF-7 and/or MDA-MB-468 cells were treated with or without 10 µM Peptide 1 for 48h. After the treatment, cells were collected and lysed. The protein levels of p53, its down-stream p21, and actin were examined by Western blotting. The Actin was used as an internal control………………………………………………………………121
Figure 3.29. MCF-7 cells were treated with 0, 10, or 20 µM of Peptide 1 or Peptide 2 for 48h. Subsequently, cells were collected, fixed with 70% ethanol, and stained with 10 µg/ml propidium iodide. The cell cycle distribution was analyzed by a flow cytometer…………………122
Figure 3.30. Table summarizing the list of interacting residues when (a) S100A1 interacts with p53 TAD and MDM2, (b) MDM2 interacts with S100A1 and p53 TAD, and (c) p53 TAD interacts with S100A1 and MDM2.123

                                

1. Mazzarello, P., A unifying concept: the history of cell theory. Nature cell biology 1999, 1 (1), E13-E15.
2. Doolittle, W. F., A paradigm gets shifty. Nature 1998, 392 (6671), 15-16.
3. Campbell, A. M., Genome organization in prokaryotes. Current opinion in genetics & development 1993, 3 (6), 837-844.
4. Pharmapproach. Major Differences between Prokaryotic and Eukaryotic Cells 2020. https://www.pharmapproach.com/differences-between-prokaryotic-and-eukaryotic-cells/.
5. Yeol, J. W.; Samarrai, W.; Barjis, I.; Ryu, Y. In Data-flow diagram representation of biological process: DNA, RNA, and protein, Proceedings of the IEEE 31st Annual Northeast Bioengineering Conference, 2005., IEEE: 2005; pp 106-107.
6. Wright, L. K.; Fisk, J. N.; Newman, D. L., DNA→ RNA: What do students think the arrow means? CBE—Life Sciences Education 2014, 13 (2), 338-348.
7. Simon, E. J. Biology The Core. https://slideplayer.com/slide/12046208/.
8. Protein Secretion (Molecular Biology). http://what-when-how.com/molecular-biology/protein-secretion-molecular-biology/.
9. Haurowitz, D. E. K. a. F. Protein. https://www.britannica.com/science/protein/Physicochemical-properties-of-the-amino-acids.
10. O’Connor, C. M.; Adams, J. U.; Fairman, J., Essentials of cell biology. Cambridge, MA: NPG Education 2010, 1, 54.
11. Bailey, R., February 11). Learn About the 4 Types of Protein Structure. https://www.thoughtco.com/protein-structure-373563.
12. Ophardt, C., E. Denaturation of Proteins. http://chemistry.elmhurst.edu/vchembook/568denaturation.html.
13. Aryal, S. Amino Acids- Properties, Structure, Classification and Functions. https://microbenotes.com/amino-acids-properties-structure-classification-and-functions/.
14. Carr, S. M. Peptide Bond formation between successive amino acids. https://www.mun.ca/biology/scarr/iGen3_06-03.html.
15. LadyofHats Main protein structures levels. https://simple.wikipedia.org/wiki/Protein_structure.
16. Moore, B. W., A soluble protein characteristic of the nervous system. Biochemical and biophysical research communications 1965, 19 (6), 739-744.
17. Moore, B. W.; McGregor, D., Chromatographic and electrophoretic fractionation of soluble proteins of brain and liver. J Biol Chem 1965, 240 (1647), 53.
18. ISOBE, T.; OKUYAMA, T., The Amino‐Acid Sequence of the α Subunit in Bovine Brain S‐100a Protein. The FEBS Journal 1981, 116 (1), 79-86.
19. Zimmer, D. B.; Eubanks, J. O.; Ramakrishnan, D.; Criscitiello, M. F., Evolution of the S100 family of calcium sensor proteins. Cell Calcium 2013, 53 (3), 170-9.
20. Marenholz, I.; Heizmann, C. W.; Fritz, G., S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochem Biophys Res Commun 2004, 322 (4), 1111-22.
21. Donato, R., S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. The international journal of biochemistry & cell biology 2001, 33 (7), 637-668.
22. Bhattacharya, S.; Chazin, W. J., Calcium-Driven Changes in S100A11 Structure Revealed. Structure 2003, 11 (7), 738-740.
23. Heizmann, C. W.; Cox, J. A., New perspectives on S100 proteins: a multi-functional Ca 2+-, Zn 2+-and Cu 2+-binding protein family. Biometals 1998, 11 (4), 383-397.
24. Santamaria-Kisiel, L.; Rintala-Dempsey, A. C.; Shaw, G. S., Calcium-dependent and -independent interactions of the S100 protein family. Biochem J 2006, 396 (2), 201-14.
25. Donato, R.; R Cannon, B.; Sorci, G.; Riuzzi, F.; Hsu, K.; J Weber, D.; L Geczy, C., Functions of S100 proteins. Current molecular medicine 2013, 13 (1), 24-57.
26. Schmidt, A. M.; Vianna, M.; Gerlach, M.; Brett, J.; Ryan, J.; Kao, J.; Esposito, C.; Hegarty, H.; Hurley, W.; Clauss, M., Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface. Journal of Biological Chemistry 1992, 267 (21), 14987-14997.
27. Neeper, M.; Schmidt, A.; Brett, J.; Yan, S.; Wang, F.; Pan, Y.; Elliston, K.; Stern, D.; Shaw, A., Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins. Journal of Biological Chemistry 1992, 267 (21), 14998-15004.
28. Dattilo, B. M.; Fritz, G.; Leclerc, E.; Kooi, C. W.; Heizmann, C. W.; Chazin, W. J., The extracellular region of the receptor for advanced glycation end products is composed of two independent structural units. Biochemistry 2007, 46 (23), 6957-70.
29. Hudson, B. I.; Carter, A. M.; Harja, E.; Kalea, A. Z.; Arriero, M.; Yang, H.; Grant, P. J.; Schmidt, A. M., Identification, classification, and expression of RAGE gene splice variants. FASEB J 2008, 22 (5), 1572-80.
30. Xie, J.; Reverdatto, S.; Frolov, A.; Hoffmann, R.; Burz, D. S.; Shekhtman, A., Structural basis for pattern recognition by the receptor for advanced glycation end products (RAGE). Journal of Biological Chemistry 2008, 283 (40), 27255-27269.
31. Xie, J.; Mendez, J. D.; Mendez-Valenzuela, V.; Aguilar-Hernandez, M. M., Cellular signalling of the receptor for advanced glycation end products (RAGE). Cell Signal 2013, 25 (11), 2185-97.
32. Hori, O.; Brett, J.; Slattery, T.; Cao, R.; Zhang, J.; Chen, J. X.; Nagashima, M.; Lundh, E. R.; Vijay, S.; Nitecki, D., The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system. Journal of Biological Chemistry 1995, 270 (43), 25752-25761.
33. Ruan, B. H.; Li, X.; Winkler, A. R.; Cunningham, K. M.; Kuai, J.; Greco, R. M.; Nocka, K. H.; Fitz, L. J.; Wright, J. F.; Pittman, D. D.; Tan, X. Y.; Paulsen, J. E.; Lin, L. L.; Winkler, D. G., Complement C3a, CpG oligos, and DNA/C3a complex stimulate IFN-alpha production in a receptor for advanced glycation end product-dependent manner. J Immunol 2010, 185 (7), 4213-22.
34. Hofmann, M. A.; Drury, S.; Fu, C.; Qu, W.; Taguchi, A.; Lu, Y.; Avila, C.; Kambham, N.; Bierhaus, A.; Nawroth, P., RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 1999, 97 (7), 889-901.
35. He, M.; Kubo, H.; Morimoto, K.; Fujino, N.; Suzuki, T.; Takahasi, T.; Yamada, M.; Yamaya, M.; Maekawa, T.; Yamamoto, Y.; Yamamoto, H., Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells. EMBO Rep 2011, 12 (4), 358-64.
36. Du Yan, S.; Chen, X.; Fu, J.; Chen, M.; Zhu, H.; Roher, A.; Slattery, T.; Zhao, L.; Nagashima, M.; Morser, J., RAGE and amyloid-β peptide neurotoxicity in Alzheimer's disease. Nature 1996, 382 (6593), 685.
37. Zhou, L. L.; Cao, W.; Xie, C.; Tian, J.; Zhou, Z.; Zhou, Q.; Zhu, P.; Li, A.; Liu, Y.; Miyata, T.; Hou, F. F.; Nie, J., The receptor of advanced glycation end products plays a central role in advanced oxidation protein products-induced podocyte apoptosis. Kidney Int 2012, 82 (7), 759-70.
38. Tesarova, P.; Cabinakova, M.; Mikulova, V.; Zima, T.; Kalousova, M., RAGE and its ligands in cancer – culprits, biomarkers, or therapeutic targets? Neoplasma 2015, 62 (03), 353-364.
39. Jing, R.-R.; Cui, M.; Sun, B.-L.; Yu, J.; Wang, H.-M., Tissue-specific expression profiling of receptor for advanced glycation end products and its soluble forms in esophageal and lung cancer. Genetic testing and molecular biomarkers 2010, 14 (3), 355-361.
40. Dahlmann, M.; Okhrimenko, A.; Marcinkowski, P.; Osterland, M.; Herrmann, P.; Smith, J.; Heizmann, C. W.; Schlag, P. M.; Stein, U., RAGE mediates S100A4-induced cell motility via MAPK/ERK and hypoxia signaling and is a prognostic biomarker for human colorectal cancer metastasis. Oncotarget 2014, 5 (10), 3220.
41. Kang, R.; Tang, D.; Lotze, M. T.; Zeh, I., Herbert J, AGER/RAGE-mediated autophagy promotes pancreatic tumorigenesis and bioenergetics through the IL6-pSTAT3 pathway. Autophagy 2012, 8 (6), 989-991.
42. Lin, L.; Zhong, K.; Sun, Z.; Wu, G.; Ding, G., Receptor for advanced glycation end products (RAGE) partially mediates HMGB1-ERKs activation in clear cell renal cell carcinoma. Journal of cancer research and clinical oncology 2012, 138 (1), 11-22.
43. Zhao, C.-B.; Bao, J.-M.; Lu, Y.-J.; Zhao, T.; Zhou, X.-H.; Zheng, D.-Y.; Zhao, S.-C., Co-expression of RAGE and HMGB1 is associated with cancer progression and poor patient outcome of prostate cancer. American journal of cancer research 2014, 4 (4), 369.
44. Zhang, S.; Hou, X.; Zi, S.; Wang, Y.; Chen, L.; Kong, B., Polymorphisms of receptor for advanced glycation end products and risk of epithelial ovarian cancer in Chinese patients. Cellular Physiology and Biochemistry 2013, 31 (4-5), 525-531.
45. Tesařová, P.; Kalousová, M.; Jachymova, M.; Mestek, O.; Petruželka, L.; Zima, T., Receptor for advanced glycation end products (RAGE)—soluble form (sRAGE) and gene polymorphisms in patients with breast cancer. Cancer investigation 2007, 25 (8), 720-725.
46. Popa, I.; Ganea, E.; Petrescu, S. M., Expression and subcellular localization of RAGE in melanoma cells. Biochemistry and Cell Biology 2014, 92 (2), 127-136.
47. Kierdorf, K.; Fritz, G., RAGE regulation and signaling in inflammation and beyond. Journal of leukocyte biology 2013, 94 (1), 55-68.
48. Padilla, L.; Dakhel, S.; Hernandez, J. L., S100 to receptor for advanced glycation end-products binding assay: looking for inhibitors. Biochem Biophys Res Commun 2014, 446 (1), 404-9.
49. Leclerc, E.; Heizmann, C., The importance of Ca2+/Zn2+ signaling S100 proteins and RAGE in translational medicine. Frontiers in bioscience (Scholar edition) 2011, 3, 1232-1262.
50. Leclerc, E.; Fritz, G.; Vetter, S. W.; Heizmann, C. W., Binding of S100 proteins to RAGE: an update. Biochim Biophys Acta 2009, 1793 (6), 993-1007.
51. Chen, H.; Xu, C.; Qing’e Jin, Z. L., S100 protein family in human cancer. American journal of cancer research 2014, 4 (2), 89.
52. Lane, D. P.; Crawford, L. V., T antigen is bound to a host protein in SY40-transformed cells. Nature 1979, 278 (5701), 261.
53. Linzer, D. I.; Levine, A. J., Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell 1979, 17 (1), 43-52.
54. Kress, M.; May, E.; Cassingena, R.; May, P., Simian virus 40-transformed cells express new species of proteins precipitable by anti-simian virus 40 tumor serum. Journal of virology 1979, 31 (2), 472-483.
55. Melero, J.; Stitt, D. T.; Mangel, W. F.; Carroll, R. B., Identification of new polypeptide species (48–55K) immunoprecipitable by antiserum to purified large T antigen and present in SV40-infected and-transformed cells. Virology 1979, 93 (2), 466-480.
56. Smith, A. E.; Smith, R.; Paucha, E., Characterization of different tumor antigens present in cells transformed by simian virus 40. Cell 1979, 18 (2), 335-346.
57. Levine, A. J.; Oren, M., The first 30 years of p53: growing ever more complex. Nature reviews cancer 2009, 9 (10), 749.
58. Oren, M.; Levine, A. J., Molecular cloning of a cDNA specific for the murine p53 cellular tumor antigen. Proceedings of the National Academy of Sciences 1983, 80 (1), 56-59.
59. Matlashewski, G.; Lamb, P.; Pim, D.; Peacock, J.; Crawford, L.; Benchimol, S., Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene. The EMBO journal 1984, 3 (13), 3257-3262.
60. Harlow, E.; Williamson, N. M.; Ralston, R.; Helfman, D. M.; Adams, T. E., Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53. Molecular and cellular biology 1985, 5 (7), 1601-1610.
61. Pennica, D.; Goeddel, D. V.; Hayflick, J. S.; Reich, N. C.; Anderson, C. W.; Levine, A. J., The amino acid sequence of murine p53 determined from a c-DNA clone. Virology 1984, 134 (2), 477-482.
62. Leppard, K.; Totty, N.; Waterfield, M.; Harlow, E.; Jenkins, J.; Crawford, L., Purification and partial amino acid sequence analysis of the cellular tumour antigen, p53, from mouse SV40‐transformed cells. The EMBO journal 1983, 2 (11), 1993-1999.
63. Zakut‐Houri, R.; Bienz‐Tadmor, B.; Givol, D.; Oren, M., Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells. The EMBO journal 1985, 4 (5), 1251-1255.
64. Wolf, D.; Harris, N.; Goldfinger, N.; Rotter, V., Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule. Molecular and cellular biology 1985, 5 (1), 127-132.
65. Wolf, D.; Rotter, V., Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences. Molecular and cellular biology 1984, 4 (7), 1402-1410.
66. Ben, Y. D.; Prideaux, V.; Chow, V.; Benchimol, S.; Bernstein, A., Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus. Oncogene 1988, 3 (2), 179-185.
67. Mowat, M.; Cheng, A.; Kimura, N.; Bernstein, A.; Benchimol, S., Rearrangements of the cellular p53 gene in erythroleukaemic cells transformed by Friend virus. Nature 1985, 314 (6012), 633.
68. Wolf, D.; Rotter, V., Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proceedings of the National Academy of Sciences 1985, 82 (3), 790-794.
69. Baker, S. J.; Fearon, E. R.; Nigro, J. M.; Preisinger, A.; Jessup, J.; Ledbetter, D.; Barker, D.; Nakamura, Y.; White, R.; Vogelstein, B., Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 1989, 244 (4901), 217-221.
70. Lane, D. P., Cancer. p53, guardian of the genome. Nature 1992, 358, 15-16.
71. Vousden, K. H.; Prives, C., Blinded by the light: the growing complexity of p53. Cell 2009, 137 (3), 413-431.
72. Joerger, A. C.; Fersht, A. R., Structural biology of the tumor suppressor p53. Annu. Rev. Biochem. 2008, 77, 557-582.
73. Vise, P. D.; Baral, B.; Latos, A. J.; Daughdrill, G. W., NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain. Nucleic acids research 2005, 33 (7), 2061-2077.
74. Dawson, R.; Müller, L.; Dehner, A.; Klein, C.; Kessler, H.; Buchner, J., The N-terminal domain of p53 is natively unfolded. Journal of molecular biology 2003, 332 (5), 1131-1141.
75. Lee, H.; Mok, K. H.; Muhandiram, R.; Park, K.-H.; Suk, J.-E.; Kim, D.-H.; Chang, J.; Sung, Y. C.; Choi, K. Y.; Han, K.-H., Local structural elements in the mostly unstructured transcriptional activation domain of human p53. Journal of Biological Chemistry 2000, 275 (38), 29426-29432.
76. Walker, K. K.; Levine, A. J., Identification of a novel p53 functional domain that is necessary for efficient growth suppression. Proceedings of the National Academy of Sciences 1996, 93 (26), 15335-15340.
77. Chang, J.; Kim, D.-H.; Lee, S. W.; Choi, K. Y.; Sung, Y. C., Transactivation ability of p53 transcriptional activation domain is directly related to the binding affinity to TATA-binding protein. Journal of Biological Chemistry 1995, 270 (42), 25014-25019.
78. Brady, C. A.; Jiang, D.; Mello, S. S.; Johnson, T. M.; Jarvis, L. A.; Kozak, M. M.; Broz, D. K.; Basak, S.; Park, E. J.; McLaughlin, M. E., Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression. Cell 2011, 145 (4), 571-583.
79. Menendez, D.; Inga, A.; Resnick, M. A., The expanding universe of p53 targets. Nature reviews Cancer 2009, 9 (10), 724.
80. Zhang, R.; Wang, H., MDM2 oncogene as a novel target for human cancer therapy. Current pharmaceutical design 2000, 6 (4), 393-416.
81. Jones, S. N.; Roe, A. E.; Donehower, L. A.; Bradley, A., Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 1995, 378 (6553), 206.
82. de Oca Luna, R. M.; Wagner, D. S.; Lozano, G., Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 1995, 378 (6553), 203.
83. Cahilly-Snyder, L.; Yang-Feng, T.; Francke, U.; George, D. L., Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line. Somatic cell and molecular genetics 1987, 13 (3), 235-244.
84. Fakharzadeh, S.; Trusko, S. P.; George, D. L., Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. The EMBO journal 1991, 10 (6), 1565-1569.
85. Nag, S.; Qin, J.; Srivenugopal, K. S.; Wang, M.; Zhang, R., The MDM2-p53 pathway revisited. Journal of biomedical research 2013, 27 (4), 254.
86. Rayburn, E. R.; Ezell, S. J.; Zhang, R., Recent advances in validating MDM2 as a cancer target. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents) 2009, 9 (8), 882-903.
87. Evans, S. C.; Viswanathan, M.; Grier, J. D.; Narayana, M.; El-Naggar, A. K.; Lozano, G., An alternatively spliced HDM2 product increases p53 activity by inhibiting HDM2. Oncogene 2001, 20 (30), 4041.
88. Perry, M. E.; Mendrysa, S. M.; Saucedo, L. J.; Tannous, P.; Holubar, M., p76MDM2 Inhibits the Ability of p90MDM2to Destabilize p53. Journal of Biological Chemistry 2000, 275 (8), 5733-5738.
89. Marine, J.-C.; Jochemsen, A. G., Mdmx as an essential regulator of p53 activity. Biochemical and biophysical research communications 2005, 331 (3), 750-760.
90. Momand, J.; Wu, H.-H.; Dasgupta, G., MDM2—master regulator of the p53 tumor suppressor protein. Gene 2000, 242 (1-2), 15-29.
91. Iwakuma, T.; Lozano, G., MDM2, an introduction. Molecular Cancer Research 2003, 1 (14), 993-1000.
92. Roth, J.; Dobbelstein, M.; Freedman, D. A.; Shenk, T.; Levine, A. J., Nucleo‐cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. The EMBO journal 1998, 17 (2), 554-564.
93. Freedman, D. A.; Levine, A. J., Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6. Molecular and cellular biology 1998, 18 (12), 7288-7293.
94. Lohrum, M. A.; Ashcroft, M.; Kubbutat, M. H.; Vousden, K. H., Identification of a cryptic nucleolar-localization signal in MDM2. Nature cell biology 2000, 2 (3), 179.
95. Argentini, M.; Barboule, N.; Wasylyk, B., The contribution of the acidic domain of MDM2 to p53 and MDM2 stability. Oncogene 2001, 20 (11), 1267.
96. Zhu, Q.; Yao, J.; Wani, G.; Wani, M. A.; Wani, A. A., Mdm2 mutant defective in binding p300 promotes ubiquitination but not degradation of p53 EVIDENCE FOR THE ROLE OF p300 In Integrating Ubiquitination and Proteolysis. Journal of Biological Chemistry 2001, 276 (32), 29695-29701.
97. Olson, D. C.; Marechal, V.; Momand, J.; Chen, J.; Romocki, C.; Levine, A., Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes. Oncogene 1993, 8 (9), 2353-2360.
98. Haupt, Y.; Maya, R.; Kazaz, A.; Oren, M., Mdm2 promotes the rapid degradation of p53. Nature 1997, 387 (6630), 296.
99. Honda, R.; Tanaka, H.; Yasuda, H., Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS letters 1997, 420 (1), 25-27.
100. Kubbutat, M. H.; Jones, S. N.; Vousden, K. H., Regulation of p53 stability by Mdm2. Nature 1997, 387 (6630), 299.
101. YANG, Y.; YU, X., Regulation of apoptosis: the ubiquitous way. The FASEB Journal 2003, 17 (8), 790-799.
102. Nakamura, S.; Roth, J. A.; Mukhopadhyay, T., Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Molecular and cellular biology 2000, 20 (24), 9391-9398.
103. Rodriguez, M. S.; Desterro, J. M.; Lain, S.; Lane, D. P.; Hay, R. T., Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation. Molecular and cellular biology 2000, 20 (22), 8458-8467.
104. Wu, X.; Bayle, J. H.; Olson, D.; Levine, A. J., The p53-mdm-2 autoregulatory feedback loop. Genes & development 1993, 7 (7a), 1126-1132.
105. Oliner, J. D.; Pietenpol, J. A.; Thiagalingam, S.; Gyuris, J.; Kinzler, K. W.; Vogelstein, B., Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature 1993, 362 (6423), 857.
106. Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.; Pavletich, N. P., Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 1996, 274 (5289), 948-953.
107. Zhao, Y.; Aguilar, A.; Bernard, D.; Wang, S., Small-molecule inhibitors of the MDM2–p53 protein–protein interaction (MDM2 Inhibitors) in clinical trials for cancer treatment: miniperspective. Journal of medicinal chemistry 2014, 58 (3), 1038-1052.
108. Thut, C. J.; Goodrich, J. A.; Tjian, R., Repression of p53-mediated transcription by MDM2: a dual mechanism. Genes & development 1997, 11 (15), 1974-1986.
109. Freedman, D.; Wu, L.; Levine, A., Functions of the MDM2 oncoprotein. Cellular and Molecular Life Sciences CMLS 1999, 55 (1), 96-107.
110. Ganguli, G.; Abecassis, J.; Wasylyk, B., MDM2 induces hyperplasia and premalignant lesions when expressed in the basal layer of the epidermis. The EMBO journal 2000, 19 (19), 5135-5147.
111. Oliner, J. D.; Saiki, A. Y.; Caenepeel, S., The role of MDM2 amplification and overexpression in tumorigenesis. Cold Spring Harbor perspectives in medicine 2016, 6 (6), a026336.
112. Jeong, H.; Kim, H. J.; Lee, S. J., Complete genome sequence of Escherichia coli strain BL21. Genome announcements 2015, 3 (2).
113. He, H.; Han, L.; Guan, W.; Li, J.; Han, W.; Yu, Y., An efficient expression and purification strategy for the production of S100 proteins in Escherichia coli. Bioengineered 2013, 4 (1), 55-58.
114. Ritchie, C., Protein purification. Iowa State University, United States. Mater Methods 2012, 2, 134.
115. Gu, T.; Gu, Y.; Zheng, Y.; Wiehl, P. E.; Kopchick, J. J., Phase separation of acetonitrile-water mixture in protein purification. Separations Technology 1994, 4 (4), 258-260.
116. Gu, Y.; Shih, P.-H., Salt-induced phase separation can effectively remove the acetonitrile from the protein sample after the preparative RP-HPLC. Enzyme and microbial technology 2004, 35 (6-7), 592-597.
117. Domon, B.; Aebersold, R., Mass spectrometry and protein analysis. science 2006, 312 (5771), 212-217.
118. El-Aneed, A.; Cohen, A.; Banoub, J., Mass spectrometry, review of the basics: electrospray, MALDI, and commonly used mass analyzers. Applied Spectroscopy Reviews 2009, 44 (3), 210-230.
119. Biswas, P., Modern Biophysical Approaches to Study Protein–Ligand Interactions. Biophysical Reviews and Letters 2018, 13 (04), 133-155.
120. Du, X.; Li, Y.; Xia, Y.-L.; Ai, S.-M.; Liang, J.; Sang, P.; Ji, X.-L.; Liu, S.-Q., Insights into protein–ligand interactions: mechanisms, models, and methods. International journal of molecular sciences 2016, 17 (2), 144.
121. Zuiderweg, E. R., Mapping protein− protein interactions in solution by NMR spectroscopy. Biochemistry 2002, 41 (1), 1-7.
122. Williamson, M. P., Using chemical shift perturbation to characterise ligand binding. Progress in nuclear magnetic resonance spectroscopy 2013, 73, 1-16.
123. Williamson, M. P., Chemical Shift Perturbation. In Modern Magnetic Resonance, Webb, G. A., Ed. Springer International Publishing: Cham, 2018; pp 995-1012.
124. De Vries, S. J.; Van Dijk, M.; Bonvin, A. M., The HADDOCK web server for data-driven biomolecular docking. Nature protocols 2010, 5 (5), 883.
125. van Zundert, G. C. P.; Rodrigues, J.; Trellet, M.; Schmitz, C.; Kastritis, P. L.; Karaca, E.; Melquiond, A. S. J.; van Dijk, M.; de Vries, S. J.; Bonvin, A., The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes. J Mol Biol 2016, 428 (4), 720-725.
126. Ngamwongsatit, P.; Banada, P. P.; Panbangred, W.; Bhunia, A. K., WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line. Journal of microbiological methods 2008, 73 (3), 211-215.
127. Berridge, M. V.; Herst, P. M.; Tan, A. S., Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology annual review 2005, 11, 127-152.
128. Wang, G.; Zhang, S.; Fernig, D. G.; Martin-Fernandez, M.; Rudland, P. S.; Barraclough, R., Mutually antagonistic actions of S100A4 and S100A1 on normal and metastatic phenotypes. Oncogene 2005, 24 (8), 1445-54.
129. Bulk, E.; Sargin, B.; Krug, U.; Hascher, A.; Jun, Y.; Knop, M.; Kerkhoff, C.; Gerke, V.; Liersch, R.; Mesters, R. M.; Hotfilder, M.; Marra, A.; Koschmieder, S.; Dugas, M.; Berdel, W. E.; Serve, H.; Muller-Tidow, C., S100A2 induces metastasis in non-small cell lung cancer. Clin Cancer Res 2009, 15 (1), 22-9.
130. Kang, M.; Lee, H. S.; Lee, Y. J.; Choi, W. S.; Park, Y. H.; Jeong, C. W.; Ku, J. H.; Kim, H. H.; Kwak, C., S100A3 suppression inhibits in vitro and in vivo tumor growth and invasion of human castration-resistant prostate cancer cells. Urology 2015, 85 (1), 273 e9-15.
131. Ochiya, T.; Takenaga, K.; Endo, H., Silencing of S100A4, a metastasis-associated protein, in endothelial cells inhibits tumor angiogenesis and growth. Angiogenesis 2014, 17 (1), 17-26.
132. Zhang, J.; Zhang, K.; Jiang, X.; Zhang, J., S100A6 as a potential serum prognostic biomarker and therapeutic target in gastric cancer. Dig Dis Sci 2014, 59 (9), 2136-44.
133. Nasser, M. W.; Qamri, Z.; Deol, Y. S.; Ravi, J.; Powell, C. A.; Trikha, P.; Schwendener, R. A.; Bai, X. F.; Shilo, K.; Zou, X.; Leone, G.; Wolf, R.; Yuspa, S. H.; Ganju, R. K., S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways. Cancer Res 2012, 72 (3), 604-15.
134. Ichikawa, M.; Williams, R.; Wang, L.; Vogl, T.; Srikrishna, G., S100A8/A9 activate key genes and pathways in colon tumor progression. Mol Cancer Res 2011, 9 (2), 133-48.
135. Grebhardt, S.; Muller-Decker, K.; Bestvater, F.; Hershfinkel, M.; Mayer, D., Impact of S100A8/A9 expression on prostate cancer progression in vitro and in vivo. J Cell Physiol 2014, 229 (5), 661-71.
136. Phipps, K. D.; Surette, A. P.; O'Connell, P. A.; Waisman, D. M., Plasminogen receptor S100A10 is essential for the migration of tumor-promoting macrophages into tumor sites. Cancer Res 2011, 71 (21), 6676-83.
137. Hao, J.; Wang, K.; Yue, Y.; Tian, T.; Xu, A.; Hao, J.; Xiao, X.; He, D., Selective expression of S100A11 in lung cancer and its role in regulating proliferation of adenocarcinomas cells. Mol Cell Biochem 2012, 359 (1-2), 323-32.
138. Zhao, F.-T.; Jia, Z.-S.; Yang, Q.; Song, L.; Jiang, X.-J., S100A14 Promotes the Growth and Metastasis of Hepatocellular Carcinoma. Asian Pacific Journal of Cancer Prevention 2013, 14 (6), 3831-3836.
139. Wang, H.; Zhang, L.; Zhang, I. Y.; Chen, X.; Da Fonseca, A.; Wu, S.; Ren, H.; Badie, S.; Sadeghi, S.; Ouyang, M.; Warden, C. D.; Badie, B., S100B promotes glioma growth through chemoattraction of myeloid-derived macrophages. Clin Cancer Res 2013, 19 (14), 3764-75.
140. Dakhel, S.; Padilla, L.; Adan, J.; Masa, M.; Martinez, J. M.; Roque, L.; Coll, T.; Hervas, R.; Calvis, C.; Messeguer, R.; Mitjans, F.; Hernandez, J. L., S100P antibody-mediated therapy as a new promising strategy for the treatment of pancreatic cancer. Oncogenesis 2014, 3, e92.
141. Todoroki, H.; Kobayashi, R.; Watanabe, M.; Minami, H.; Hidaka, H., Purification, characterization, and partial sequence analysis of a newly identified EF-hand type 13-kDa Ca (2+)-binding protein from smooth muscle and non-muscle tissues. Journal of Biological Chemistry 1991, 266 (28), 18668-18673.
142. Ohta, H.; Sasaki, T.; Naka, M.; Hiraoka, O.; Miyamoto, C.; Furuichi, Y.; Tanaka, T., Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle. FEBS letters 1991, 295 (1-3), 93-96.
143. Inada, H.; Naka, M.; Tanaka, T.; Davey, G. E.; Heizmann, C. W., Human S100A11 exhibits differential steady-state RNA levels in various tissues and a distinct subcellular localization. Biochemical and biophysical research communications 1999, 263 (1), 135-138.
144. Sakaguchi, M.; Sonegawa, H.; Murata, H.; Kitazoe, M.; Futami, J.-i.; Kataoka, K.; Yamada, H.; Huh, N.-h., S100A11, an dual mediator for growth regulation of human keratinocytes. Molecular biology of the cell 2008, 19 (1), 78-85.
145. Tian, T.; Hao, J.; Xu, A.; Hao, J.; Luo, C.; Liu, C.; Huang, L.; Xiao, X.; He, D., Determination of metastasis-associated proteins in non-small cell lung cancer by comparative proteomic analysis. Cancer Sci 2007, 98 (8), 1265-74.
146. Wang, G.; Wang, X.; Wang, S.; Song, H.; Sun, H.; Yuan, W.; Cao, B.; Bai, J.; Fu, S., Colorectal cancer progression correlates with upregulation of S100A11 expression in tumor tissues. Int J Colorectal Dis 2008, 23 (7), 675-82.
147. Fan, Ca2+-binding protein S100A11: A novel diagnostic marker for breast carcinoma. Oncology Reports 2010, 23 (5).
148. Ohuchida, K.; Mizumoto, K.; Ohhashi, S.; Yamaguchi, H.; Konomi, H.; Nagai, E.; Yamaguchi, K.; Tsuneyoshi, M.; Tanaka, M., S100A11, a putative tumor suppressor gene, is overexpressed in pancreatic carcinogenesis. Clin Cancer Res 2006, 12 (18), 5417-22.
149. Xiao, M. B.; Jiang, F.; Ni, W. K.; Chen, B. Y.; Lu, C. H.; Li, X. Y.; Ni, R. Z., High expression of S100A11 in pancreatic adenocarcinoma is an unfavorable prognostic marker. Med Oncol 2012, 29 (3), 1886-91.
150. Meding, S.; Balluff, B.; Elsner, M.; Schone, C.; Rauser, S.; Nitsche, U.; Maak, M.; Schafer, A.; Hauck, S. M.; Ueffing, M.; Langer, R.; Hofler, H.; Friess, H.; Rosenberg, R.; Walch, A., Tissue-based proteomics reveals FXYD3, S100A11 and GSTM3 as novel markers for regional lymph node metastasis in colon cancer. J Pathol 2012, 228 (4), 459-70.
151. Wang, C.; Zhang, Z.; Li, L.; Zhang, J.; Wang, J.; Fan, J.; Jiao, B.; Zhao, S., S100A11 is a migration-related protein in laryngeal squamous cell carcinoma. Int J Med Sci 2013, 10 (11), 1552-9.
152. Anania, M. C.; Miranda, C.; Vizioli, M. G.; Mazzoni, M.; Cleris, L.; Pagliardini, S.; Manenti, G.; Borrello, M. G.; Pierotti, M. A.; Greco, A., S100A11 overexpression contributes to the malignant phenotype of papillary thyroid carcinoma. J Clin Endocrinol Metab 2013, 98 (10), E1591-600.
153. Memon, A. A.; Sorensen, B. S.; Meldgaard, P.; Fokdal, L.; Thykjaer, T.; Nexo, E., Down-regulation of S100C is associated with bladder cancer progression and poor survival. Clinical cancer research 2005, 11 (2), 606-611.
154. Liu, Y.; Han, X.; Gao, B., Knockdown of S100A11 expression suppresses ovarian cancer cell growth and invasion. Exp Ther Med 2015, 9 (4), 1460-1464.
155. Baudier, J.; Glasser, N.; Gerard, D., Ions binding to S100 proteins. I. Calcium-and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein. Journal of Biological Chemistry 1986, 261 (18), 8192-8203.
156. Amburgey, J. C.; Abildgaard, F.; Starich, M. R.; Shah, S.; Hilt, D. C.; Weber, D. J., 1 H, 13 C and 15 N NMR assignments and solution secondary structure of rat Apo-S100β. Journal of biomolecular NMR 1995, 6 (2), 171-179.
157. Drohat, A. C.; Amburgey, J. C.; Abildgaard, F.; Starich, M. R.; Baldisseri, D.; Weber, D. J., Solution structure of rat apo-S100B (ββ) as determined by NMR spectroscopy. Biochemistry 1996, 35 (36), 11577-11588.
158. Rickmann, M.; Wolff, J., S100 protein expression in subpopulations of neurons of rat brain. Neuroscience 1995, 67 (4), 977-991.
159. Sorci, G.; Bianchi, R.; Giambanco, I.; Rambotti, M.; Donato, R., Replicating myoblasts and fused myotubes express the calcium-regulated proteins S100A1 and S100B. Cell Calcium 1999, 25 (2), 93-106.
160. Rambotti, M.; Giambanco, I.; Spreca, A.; Donato, R., S100B and S100A1 proteins in bovine retina: their calcium-dependent stimulation of a membrane-bound guanylate cyclase activity as investigated by ultracytochemistry. Neuroscience 1999, 92 (3), 1089-1101.
161. Arcuri, C.; Giambanco, I.; Bianchi, R.; Donato, R., Annexin V, annexin VI, S100A1 and S100B in developing and adult avian skeletal muscles. Neuroscience 2002, 109 (2), 371-388.
162. Vives, V.; Alonso, G.; Solal, A. C.; Joubert, D.; Legraverend, C., Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice. J Comp Neurol 2003, 457 (4), 404-19.
163. Deloulme, J. C.; Raponi, E.; Gentil, B. J.; Bertacchi, N.; Marks, A.; Labourdette, G.; Baudier, J., Nuclear expression of S100B in oligodendrocyte progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation. Mol Cell Neurosci 2004, 27 (4), 453-65.
164. Hachem, S.; Aguirre, A.; Vives, V.; Marks, A.; Gallo, V.; Legraverend, C., Spatial and temporal expression of S100B in cells of oligodendrocyte lineage. Glia 2005, 51 (2), 81-97.
165. Heizmann, C. W.; Fritz, G.; Schafer, B., S100 proteins: structure, functions and pathology. Front Biosci 2002, 7 (1), 1356-1368.
166. Donato, R., Intracellular and extracellular roles of S100 proteins. Microsc Res Tech 2003, 60 (6), 540-51.
167. Ostendorp, T.; Leclerc, E.; Galichet, A.; Koch, M.; Demling, N.; Weigle, B.; Heizmann, C. W.; Kroneck, P. M.; Fritz, G., Structural and functional insights into RAGE activation by multimeric S100B. The EMBO journal 2007, 26 (16), 3868-3878.
168. Park, H.; Boyington, J. C., The 1.5 Å crystal structure of human receptor for advanced glycation endproducts (RAGE) ectodomains reveals unique features determining ligand binding. Journal of Biological Chemistry 2010, jbc. M110. 169276.
169. Donato, R.; Sorci, G.; Riuzzi, F.; Arcuri, C.; Bianchi, R.; Brozzi, F.; Tubaro, C.; Giambanco, I., S100B's double life: intracellular regulator and extracellular signal. Biochim Biophys Acta 2009, 1793 (6), 1008-22.
170. Nash, D. L.; Bellolio, M. F.; Stead, L. G., S100 as a marker of acute brain ischemia: a systematic review. Neurocrit Care 2008, 8 (2), 301-7.
171. Dagdan, E.; Morris, D. W.; Campbell, M.; Hill, M.; Rothermundt, M.; Kastner, F.; Hohoff, C.; von Eiff, C.; Krakowitzky, P.; Gill, M.; McKeon, P.; Roche, S., Functional assessment of a promoter polymorphism in S100B, a putative risk variant for bipolar disorder. Am J Med Genet B Neuropsychiatr Genet 2011, 156B (6), 691-9.
172. Egberts, F.; Pollex, A.; Egberts, J.-H.; Kaehler, K. C.; Weichenthal, M.; Hauschild, A., Long-term survival analysis in metastatic melanoma: serum S100B is an independent prognostic marker and superior to LDH. Oncology Research and Treatment 2008, 31 (7), 380-384.
173. Weide, B.; Richter, S.; Buttner, P.; Leiter, U.; Forschner, A.; Bauer, J.; Held, L.; Eigentler, T. K.; Meier, F.; Garbe, C., Serum S100B, lactate dehydrogenase and brain metastasis are prognostic factors in patients with distant melanoma metastasis and systemic therapy. PLoS One 2013, 8 (11), e81624.
174. Rouhiainen, A.; Kuja-Panula, J.; Tumova, S.; Rauvala, H., RAGE-mediated cell signaling. In Calcium-Binding Proteins and RAGE, Springer: 2013; pp 239-263.
175. Saleh, A.; Smith, D. R.; Tessler, L.; Mateo, A. R.; Martens, C.; Schartner, E.; Van der Ploeg, R.; Toth, C.; Zochodne, D. W.; Fernyhough, P., Receptor for advanced glycation end-products (RAGE) activates divergent signaling pathways to augment neurite outgrowth of adult sensory neurons. Experimental neurology 2013, 249, 149-159.
176. Huang, Y. K.; Chou, R. H.; Yu, C., Tranilast Blocks the Interaction between the Protein S100A11 and Receptor for Advanced Glycation End Products (RAGE) V Domain and Inhibits Cell Proliferation. J Biol Chem 2016, 291 (27), 14300-10.
177. Bianchi, R.; Giambanco, I.; Arcuri, C.; Donato, R., Subcellular localization of S100A11 (S100C) in LLC‐PK1 renal cells: Calcium‐and protein kinase c–dependent association of S100A11 with S100B and vimentin intermediate filaments. Microscopy research and technique 2003, 60 (6), 639-651.
178. Deloulme, J. C.; Assard, N.; Mbele, G. O.; Mangin, C.; Kuwano, R.; Baudier, J., S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo. J Biol Chem 2000, 275 (45), 35302-10.
179. Hung, K. W.; Chang, Y. M.; Yu, C., NMR structure note: the structure of human calcium-bound S100A11. J Biomol NMR 2012, 54 (2), 211-5.
180. Smith, S. P.; Shaw, G. S., A novel calcium-sensitive switch revealed by the structure of human S100B in the calcium-bound form. Structure 1998, 6 (2), 211-222.
181. Gupta, A. A.; Chou, R. H.; Li, H.; Yang, L. W.; Yu, C., Structural insights into the interaction of human S100B and basic fibroblast growth factor (FGF2): Effects on FGFR1 receptor signaling. Biochim Biophys Acta 2013, 1834 (12), 2606-19.
182. Baudier, J.; Gerard, D., Ions binding to S100 proteins. II. Conformational studies and calcium-induced conformational changes in S100 alpha alpha protein: the effect of acidic pH and calcium incubation on subunit exchange in S100a (alpha beta) protein. Journal of Biological Chemistry 1986, 261 (18), 8204-8212.
183. Pröpper, C.; Huang, X.; Roth, J.; Sorg, C.; Nacken, W., Analysis of the MRP8-MRP14 protein-protein interaction by the two-hybrid system suggests a prominent role of the C-terminal domain of S100 proteins in dimer formation. Journal of Biological Chemistry 1999, 274 (1), 183-188.
184. Goddard, T.; Kneller, D., SPARKY 3. University of California, San Francisco 2008.
185. de Vries, S. J.; van Dijk, M.; Bonvin, A. M., The HADDOCK web server for data-driven biomolecular docking. Nat Protoc 2010, 5 (5), 883-97.
186. Dominguez, C.; Boelens, R.; Bonvin, A. M., HADDOCK: a protein− protein docking approach based on biochemical or biophysical information. Journal of the American Chemical Society 2003, 125 (7), 1731-1737.
187. Hubbard, S. J.; Thornton, J. M., Naccess. Computer Program, Department of Biochemistry and Molecular Biology, University College London 1993, 2 (1).
188. Linge, J. P.; Williams, M. A.; Spronk, C. A.; Bonvin, A. M.; Nilges, M., Refinement of protein structures in explicit solvent. Proteins: Structure, Function, and Bioinformatics 2003, 50 (3), 496-506.
189. DeLano, W. L., Pymol: An open-source molecular graphics tool. CCP4 Newsletter On Protein Crystallography 40, 82-92.
190. Takeuchi, K.; Wagner, G., NMR studies of protein interactions. Curr Opin Struct Biol 2006, 16 (1), 109-17.
191. Williamson, M. P., Using chemical shift perturbation to characterise ligand binding. Prog Nucl Magn Reson Spectrosc 2013, 73, 1-16.
192. Laskowski, R. A.; Rullmann, J. A. C.; MacArthur, M. W.; Kaptein, R.; Thornton, J. M., AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. Journal of biomolecular NMR 1996, 8 (4), 477-486.
193. Sparvero, L. J.; Asafu-Adjei, D.; Kang, R.; Tang, D.; Amin, N.; Im, J.; Rutledge, R.; Lin, B.; Amoscato, A. A.; Zeh, H. J.; Lotze, M. T., RAGE (Receptor for Advanced Glycation Endproducts), RAGE ligands, and their role in cancer and inflammation. J Transl Med 2009, 7, 17.
194. Donato, R., RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins. Current molecular medicine 2007, 7 (8), 711-724.
195. Cecil, D. L.; Johnson, K.; Rediske, J.; Lotz, M.; Schmidt, A. M.; Terkeltaub, R., Inflammation-Induced Chondrocyte Hypertrophy Is Driven by Receptor for Advanced Glycation End Products. The Journal of Immunology 2005, 175 (12), 8296-8302.
196. Cecil, D. L.; Terkeltaub, R., Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes. The Journal of Immunology 2008, 180 (12), 8378-8385.
197. Yan, S. S.; Wu, Z.-Y.; Zhang, H. P.; Furtado, G.; Chen, X.; Yan, S. F.; Schmidt, A. M.; Brown, C.; Stern, A.; Lafaille, J., Suppression of experimental autoimmune encephalomyelitis by selective blockade of encephalitogenic T-cell infiltration of the central nervous system. Nature medicine 2003, 9 (3), 287-293.
198. Javid, J.; Mir, R.; Julka, P.; Ray, P.; Saxena, A., Association of p53 and mdm2 in the development and progression of non-small cell lung cancer. Tumor Biology 2015, 36 (7), 5425-5432.
199. McEvoy, J.; Ulyanov, A.; Brennan, R.; Wu, G.; Pounds, S.; Zhang, J.; Dyer, M. A., Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma. PloS one 2012, 7 (8), e42739.
200. Momand, J.; Jung, D.; Wilczynski, S.; Niland, J., The MDM2 gene amplification database. Nucleic acids research 1998, 26 (15), 3453-3459.
201. Weaver, J.; Downs-Kelly, E.; Goldblum, J. R.; Turner, S.; Kulkarni, S.; Tubbs, R. R.; Rubin, B. P.; Skacel, M., Fluorescence in situ hybridization for MDM2 gene amplification as a diagnostic tool in lipomatous neoplasms. Modern Pathology 2008, 21 (8), 943.
202. Weaver, J.; Goldblum, J. R.; Turner, S.; Tubbs, R. R.; Wang, W.-L.; Lazar, A. J.; Rubin, B. P., Detection of MDM2 gene amplification or protein expression distinguishes sclerosing mesenteritis and retroperitoneal fibrosis from inflammatory well-differentiated liposarcoma. Modern Pathology 2009, 22 (1), 66.
203. Landers, J. E.; Cassel, S. L.; George, D. L., Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein. Cancer research 1997, 57 (16), 3562-3568.
204. Shvarts, A.; Steegenga, W.; Riteco, N.; Van Laar, T.; Dekker, P.; Bazuine, M.; Van Ham, R.; Van Oordt, W. V. D. H.; Hateboer, G.; Van der Eb, A., MDMX: a novel p53‐binding protein with some functional properties of MDM2. The EMBO journal 1996, 15 (19), 5349-5357.
205. Shvarts, A.; Bazuine, M.; Dekker, P.; Ramos, Y. F.; Steegenga, W. T.; Merckx, G.; Ham, R. v.; Houven van Oordt, W. v. d.; van der Eb, A. J.; Jochemsen, A., Isolation and identification of the human homolog of a new p53-binding protein, Mdmx. 1997.
206. Böttger, V.; Böttger, A.; Garcia-Echeverria, C.; Ramos, Y. F.; van der Eb, A. J.; Jochemsen, A. G.; Lane, D. P., Comparative study of the p53-mdm2 and p53-MDMX interfaces. Oncogene 1999, 18 (1), 189-199.
207. Toledo, F.; Wahl, G. M., MDM2 and MDM4: p53 regulators as targets in anticancer therapy. The international journal of biochemistry & cell biology 2007, 39 (7-8), 1476-1482.
208. Kawai, H.; Wiederschain, D.; Yuan, Z.-M., Critical contribution of the MDM2 acidic domain to p53 ubiquitination. Molecular and cellular biology 2003, 23 (14), 4939-4947.
209. Meulmeester, E.; Frenk, R.; Stad, R.; De Graaf, P.; Marine, J.-C.; Vousden, K. H.; Jochemsen, A. G., Critical role for a central part of Mdm2 in the ubiquitylation of p53. Molecular and cellular biology 2003, 23 (14), 4929-4938.
210. Chène, P.; Fuchs, J.; Carena, I.; Furet, P.; Garcı́a Echeverrı́a, C., Study of the cytotoxic effect of a peptidic inhibitor of the p53–hdm2 interaction in tumor cells. FEBS letters 2002, 529 (2-3), 293-297.
211. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C., In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004, 303 (5659), 844-848.
212. Wang, S.; Zhao, Y.; Aguilar, A.; Bernard, D.; Yang, C.-Y., Targeting the MDM2–p53 protein–protein interaction for new cancer therapy: progress and challenges. Cold Spring Harbor perspectives in medicine 2017, 7 (5), a026245.
213. Shangary, S.; Ding, K.; Qiu, S.; Nikolovska-Coleska, Z.; Bauer, J. A.; Liu, M.; Wang, G.; Lu, Y.; McEachern, D.; Bernard, D., Reactivation of p53 by a specific MDM2 antagonist (MI-43) leads to p21-mediated cell cycle arrest and selective cell death in colon cancer. Molecular cancer therapeutics 2008, 7 (6), 1533-1542.
214. Shangary, S.; Qin, D.; McEachern, D.; Liu, M.; Miller, R. S.; Qiu, S.; Nikolovska-Coleska, Z.; Ding, K.; Wang, G.; Chen, J., Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. Proceedings of the National Academy of Sciences 2008, 105 (10), 3933-3938.
215. Hu, B.; Gilkes, D. M.; Chen, J., Efficient p53 activation and apoptosis by simultaneous disruption of binding to MDM2 and MDMX. Cancer Research 2007, 67 (18), 8810-8817.
216. Shiheido, H.; Takashima, H.; Doi, N.; Yanagawa, H., mRNA display selection of an optimized MDM2-binding peptide that potently inhibits MDM2-p53 interaction. PloS one 2011, 6 (3).
217. Schäfer, B. W.; Heizmann, C. W., The S100 family of EF-hand calcium-binding proteins: functions and pathology. Trends in biochemical sciences 1996, 21 (4), 134-140.
218. Fernandez-Fernandez, M. R.; Rutherford, T. J.; Fersht, A. R., Members of the S100 family bind p53 in two distinct ways. Protein Sci 2008, 17 (10), 1663-70.
219. van Dieck, J.; Fernandez-Fernandez, M. R.; Veprintsev, D. B.; Fersht, A. R., Modulation of the oligomerization state of p53 by differential binding of proteins of the S100 family to p53 monomers and tetramers. Journal of Biological Chemistry 2009, 284 (20), 13804-13811.
220. van Dieck, J.; Teufel, D. P.; Jaulent, A. M.; Fernandez-Fernandez, M. R.; Rutherford, T. J.; Wyslouch-Cieszynska, A.; Fersht, A. R., Posttranslational modifications affect the interaction of S100 proteins with tumor suppressor p53. Journal of molecular biology 2009, 394 (5), 922-930.
221. van Dieck, J.; Lum, J. K.; Teufel, D. P.; Fersht, A. R., S100 proteins interact with the N-terminal domain of MDM2. FEBS letters 2010, 584 (15), 3269-3274.
222. Nagahara, H.; Vocero-Akbani, A. M.; Snyder, E. L.; Ho, A.; Latham, D. G.; Lissy, N. A.; Becker-Hapak, M.; Ezhevsky, S. A.; Dowdy, S. F., Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27 Kip1 induces cell migration. Nature medicine 1998, 4 (12), 1449.
223. Fischer, P. M.; Krausz, E.; Lane, D. P., Cellular delivery of impermeable effector molecules in the form of conjugates with peptides capable of mediating membrane translocation. Bioconjugate chemistry 2001, 12 (6), 825-841.
224. Borcherds, W.; Theillet, F.-X.; Katzer, A.; Finzel, A.; Mishall, K. M.; Powell, A. T.; Wu, H.; Manieri, W.; Dieterich, C.; Selenko, P., Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells. Nature chemical biology 2014, 10 (12), 1000.
225. Gupta, A. A.; Mohan, S. K.; Chin, Y., 1 H, 13 C and 15 N backbone and side chain resonance assignments of human halo S100A1. Biomolecular NMR assignments 2012, 6 (2), 213-215.
226. Schrödinger, L., The PyMOL Molecular Graphics System, Version 1.5. 0.1. Schrödinger, LLC New York: 2010.
227. Liu, X.; Obianyo, O.; Chan, C. B.; Huang, J.; Xue, S.; Yang, J. J.; Zeng, F.; Goodman, M.; Ye, K., Biochemical and biophysical investigation of the brain-derived neurotrophic factor mimetic 7, 8-dihydroxyflavone in the binding and activation of the TrkB receptor. Journal of Biological Chemistry 2014, 289 (40), 27571-27584.
228. Stoll, R.; Renner, C.; Mühlhahn, P.; Hansen, S.; Schumacher, R.; Hesse, F.; Kaluza, B.; Engh, R. A.; Voelter, W.; Holak, T. A., Letter to the Editor: Sequence-specific 1H, 15N, and 13C assignment of the N-terminal domain of the human oncoprotein MDM2 that binds to p53. Journal of biomolecular NMR 2000, 17 (1), 91-92.
229. Chi, S.-W.; Lee, S.-H.; Kim, D.-H.; Ahn, M.-J.; Kim, J.-S.; Woo, J.-Y.; Torizawa, T.; Kainosho, M.; Han, K.-H., Structural details on mdm2-p53 interaction. Journal of Biological Chemistry 2005, 280 (46), 38795-38802.
230. Wong, T. S.; Rajagopalan, S.; Freund, S. M.; Rutherford, T. J.; Andreeva, A.; Townsley, F. M.; Petrovich, M.; Fersht, A. R., Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53. Nucleic acids research 2009, 37 (20), 6765-6783.
231. Joliot, A.; Prochiantz, A., Transduction peptides: from technology to physiology. Nature cell biology 2004, 6 (3), 189.
232. Snyder, E. L.; Meade, B. R.; Saenz, C. C.; Dowdy, S. F., Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide. PLoS biology 2004, 2 (2), e36.
233. Deshayes, S.; Morris, M.; Divita, G.; Heitz, F., Cell-penetrating peptides: tools for intracellular delivery of therapeutics. Cellular and Molecular Life Sciences CMLS 2005, 62 (16), 1839-1849.
234. Brooks, H.; Lebleu, B.; Vivès, E., Tat peptide-mediated cellular delivery: back to basics. Advanced drug delivery reviews 2005, 57 (4), 559-577.
235. Ruben, S.; Perkins, A.; Purcell, R.; Joung, K.; Sia, R.; Burghoff, R.; Haseltine, W.; Rosen, C., Structural and functional characterization of human immunodeficiency virus tat protein. Journal of virology 1989, 63 (1), 1-8.
236. Fawell, S.; Seery, J.; Daikh, Y.; Moore, C.; Chen, L. L.; Pepinsky, B.; Barsoum, J., Tat-mediated delivery of heterologous proteins into cells. Proceedings of the National Academy of Sciences 1994, 91 (2), 664-668.
237. Vives, E.; Brodin, P.; Lebleu, B., A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. Journal of Biological Chemistry 1997, 272 (25), 16010-16017.
238. Loret, E. P.; Georgel, P.; Johnson, W. C.; Ho, P. S., Circular dichroism and molecular modeling yield a structure for the complex of human immunodeficiency virus type 1 trans-activation response RNA and the binding region of Tat, the trans-acting transcriptional activator. Proceedings of the National Academy of Sciences 1992, 89 (20), 9734-9738.
239. Greenfield, N. J.; Fasman, G. D., Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 1969, 8 (10), 4108-4116.
240. Luan, L.; Meng, Q.; Xu, L.; Meng, Z.; Yan, H.; Liu, K., Peptide amphiphiles with multifunctional fragments promoting cellular uptake and endosomal escape as efficient gene vectors. Journal of Materials Chemistry B 2015, 3 (6), 1068-1078.
241. Royer, C. A., Probing protein folding and conformational transitions with fluorescence. Chemical reviews 2006, 106 (5), 1769-1784.
242. Leroy, B.; Girard, L.; Hollestelle, A.; Minna, J. D.; Gazdar, A. F.; Soussi, T., Analysis of TP 53 Mutation Status in Human Cancer Cell Lines: A Reassessment. Human mutation 2014, 35 (6), 756-765.
243. Sutherland, R. L.; Hall, R. E.; Taylor, I. W., Cell proliferation kinetics of MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on exponentially growing and plateau-phase cells. Cancer research 1983, 43 (9), 3998-4006.
244. Aroui, S.; Brahim, S.; De Waard, M.; Kenani, A., Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: a comparative study. Biochemical and biophysical research communications 2010, 391 (1), 419-425.
245. Fischer, R.; Köhler, K.; Fotin-Mleczek, M.; Brock, R., A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. Journal of Biological Chemistry 2004, 279 (13), 12625-12635.
246. Yu, Y.-L.; Chou, R.-H.; Liang, J.-H.; Chang, W.-J.; Su, K.-J.; Tseng, Y.-J.; Huang, W.-C.; Wang, S.-C.; Hung, M.-C., Targeting the EGFR/PCNA signaling suppresses tumor growth of triple-negative breast cancer cells with cell-penetrating PCNA peptides. PLoS One 2013, 8 (4).
247. Nagata, T.; Shirakawa, K.; Kobayashi, N.; Shiheido, H.; Tabata, N.; Sakuma-Yonemura, Y.; Horisawa, K.; Katahira, M.; Doi, N.; Yanagawa, H., Structural basis for inhibition of the MDM2: p53 interaction by an optimized MDM2-binding peptide selected with mRNA display. PloS one 2014, 9 (10), e109163.

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